Cryo-tomography reveals rigid-body motion and organization of apicomplexan invasion machinery

The apical complex is a specialized collection of cytoskeletal and secretory machinery in apicomplexan parasites, which include the pathogens that cause malaria and toxoplasmosis. Its structure and mechanism of motion are poorly understood. We used cryo-FIB-milling and cryo-electron tomography to visualize the 3D-structure of the apical complex in its protruded and retracted states. Averages of conoid-fibers revealed their polarity and unusual nine-protofilament arrangement with associated proteins connecting and likely stabilizing the fibers. Neither the structure of the conoid-fibers nor the architecture of the spiral-shaped conoid complex change during protrusion or retraction. Thus, the conoid moves as a rigid body, and is not spring-like and compressible, as previously suggested. Instead, the apical-polar-rings (APR), previously considered rigid, dilate during conoid protrusion. We identified actin-like filaments connecting the conoid and APR during protrusion, suggesting a role during conoid movements. Furthermore, our data capture the parasites in the act of secretion during conoid protrusion.


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In this study, 167 tilt-series were collected from the cryo-FIB milled lamella of native parasites and 20 of the reconstructed tomograms contained the apical complex (13 in the protruded, and 7 in the retracted state). 1160 and 721 8-nm conoid fiber repeats were selected from the protruded and retracted native conoid tomograms, respectively. For the PCR repeating structure, 180 and 159 repeats of P2 and P3 were selected for subtomogram averaging, respectively. Additionally, 19 tilt-series were recorded of the apical region of detergent-treated (not cryo-FIB milled) parasites and 386 8-nm ICMT repeats were picked from the reconstructed tomograms of detergent-extracted samples. 25 subtomograms of the microneme tip were extracted, aligned and averaged. All 167 tilt series collected on the electron microscope were analyzed and we determined these to be sufficient owing to low observed variability. All studies must disclose on these points even when the disclosure is negative.

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No data were excluded All the parasite specimen in this work were prepared from multiple rounds of cell culture and sample freezing (n>3). The lamellae of the specimen were milled over more than 20 sessions and the tilt series were collected during 18 cryo-EM data acquisition sessions at different times. All experimental findings were reliably reproduced with consistent results.
Parasites were allocated randomly into experimental groups to either prepared in an intracellular-like buffer or incubated with 10 !m calcium ionophore so that the majority of parasites have conoids in the retracted or protruded states, respectively The persons performing cryo-FIB milling and EM imaging were unaware of the sample identity. No blinding effort was taken during data analysis.
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